Retinoic acid (RA), the most potent biologically active form of vitamin A, plays an important role during growth, differentiation, immune response, reproduction, and embryonic development. Both maternal insuffiency and maternal excess of vitamin A are associated with developmental abnormalities. RA treatment of P19 embryonal carcinoma cells causes differentiation to either endodermal or neuronal cells, depending on the culture conditions. Pre-B cell leukemia transcription factor 1 (PBX1), PBX2 and PBX3 mRNA levels, and PBX1/2/3 protein levels are elevated upon treatment of P19 cells with RA. PBX proteins function as dimeric partners with several HOX proteins mediating gene expression during development. This RA-dependent increase in PBX1/2/3 expression has been demonstrated to be critical for differentiation of P19 cells to both endodermal and neuronal cells. In addition, the expression of two genes, bone morphogenetic protein 4 (BMP4) and decornin (DCN) have been shown to require RA-dependent increase in PBX1/2/3 expression during endodermal cell differentiation. The goal of the proposed studies is to elucidation the role of this RA-dependent increase in PBX1/2/3 levels during differentiation of P19 cells to endodermal and neuronal cells. We therefore plan: (1) to further characterize the role of PBX172/3 proteins during differentiation of P19 cells to endodermal, neuronal and cardiac cells; (2) to determine if induction of BMP4 and/or DCN expression by PBX1/2/3 proteins is required for RA-dependent differentiation of P19 cells to endodermal and/or neuronal cells; and (3) to identify and characterize additional PBX1/2/3-regulated genes during RA-dependent differentiation of P19 cells to endodermal and neuronal cells. These studies will further the understanding of the role of PBX during mammalian development and further elucidate the details of one RA-dependent pathway of signaling during differentiation.